Gastrin is the major gastrointestinal hormone known to regulate both acid secretion and gastrointestinal cell growth. Pathologic states such as atrophic gastritis and Helicobacter pylori infection correlate with elevated serum gastrin levels and subsequently with the development of peptic disorders. As a results of gastrin's growth promoting effects, hypergastrinemic states including gastinomas are a permissive environment for neoplastic transformation in the stomach and arguably in the colon. Therefore the long term objective of this project is to understand how the gastrin gene is regulated at the transcriptional level by extracellular mediator and cell specific signals. We have successfully identified an element called gERE that confers EGF and phorbol ester responsiveness to the gastrin promoter. In addition to demonstrating that a transcriptional activator, i.e., Sp1, binds to gERE, we have recently cloned a transcriptional repressor, ZBP-89, that competes with Sp1 for binding to gERE. ZBP represses basal promoter activity and blocks EGF induction of the gastrin promoter. Therefore the specific aims of this application are a) to examine the inducible expression of the human gastrin gene in human gastric cell lines and b) to study the signaling pathways relevant to induction of the gastrin promoter via gERE. Inducible regulation of the gastrin promoter by Spl and ZBP-89 will be tested in human stomach cell lines by cotransfecting these expression vectors with gastrin reporter constructs. ZBP-89 is a novel factor who functional domains will be mapped by site-directed mutagenesis. Whether ras-dependent or independent pathways mediate gastrin promoter activity via the gERE element will be tested using ras dominant negative expression vectors. In addition, the regulation of SP1 and ZBP-89 by phosphorylation in response to EGF induction will be studied by immunoprecipitation of these factors from nuclear extracts. Moreover, the peptide domain phsoophorylated by whole cell extracts from stimulated and stimulated cells will be studied using digests of the cloned GST-Spl or GST-ZBP-89 fusion proteins for a solid phase kinase assay and compared to the in vivo phosphorylation pattern of the endogenous proteins. Taken together, these studies will further understanding of how the human gastrin gene is regulated in human gastric cells. Further, the identification of a transcriptional repressor and how it inhibits gastrin promoter activation may direct the design of new therapies that block gastrin synthesis and perhaps the pathologic consequences of hyperfastrinemia, e.g. neoplastic transformation.